Alkaloid that inhibits biosynthesis of mycotoxins and method for screening for mycotoxin inhibitors

ABSTRACT

The present invention provides an alkaloid compound that is an inhibitor of mycotoxin biosynthesis. The alkaloid is an alkenyl piperidine amide wherein the alkenyl is a C18 alkenyl with one or more double bonds. The alkaloid inhibits transcription of the fungus genes nor-1, tri5, and ipnA. The present invention further provides a method for identifying compounds, which inhibit biosynthesis of aflatoxin in  Aspergillus  spp. and deoxynivalenol in  Gibberella  spp. without inhibiting growth of the fungus in vitro.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional application Ser.No. 60/156,381, which was filed Sep. 28, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to an alkaloid compound that inhibitsbiosynthesis of particular products of secondary metabolism. Inparticular, the present invention relates to an alkenyl piperidine amidewherein the alkenyl is a C18 alkenyl with one or more double bonds,which can be isolated from Piper nigrum, that inhibits transcription offungus genes nor-1, tri5, ver-1, verA, fas-1a, omt-1, alfR and ipnA. Thepresent invention further relates to a method for identifying compoundsthat inhibit the biosynthesis of mycotoxins in fungi. In particular, amethod for identifying compounds that inhibit biosynthesis of aflatoxinin Aspergillus spp. and deoxynivalenol in Gibberella spp.

(2) Description of Related Art

Mycotoxins are a group of structurally heterogeneous secondarymetabolites produced by a diverse group of fungal plants pathogens.Infestation of crops and food commodities by mycotoxin producing fungiis a serious problem in view of the immunosuppressive, carcinogenic,cytotoxic, and teratogenic effects of the compounds in humans andanimals. One of the most economically important mycotoxins worldwide isaflatoxin, a polyketide produced by several Aspergillus spp. Aflatoxinis the best studied of the mycotoxins and much of the molecular biologyof the biosynthetic pathway has been determined in Aspergillus flavus,Aspergillus parasiticus, and Aspergillus nidulans. Aspergillus flavusproduces aflatoxin B1 and aflatoxin B2 whereas Aspergillus parasiticusproduces in addition aflatoxin G1 and G2. Aspergillus nidulans, which isnot considered to be an agricultural threat, has been used as a modelgenetic system for studies of aflatoxin biosynthesis because it producessterigmatocystin, an aflatoxin precursor. The genes for aflatoxinbiosynthesis are clustered in all three species. The molecular biologyof aflatoxin biosynthesis is reviewed by Trail et al., in Microbiol.141: 755-765 (1995). Aspergillus flavus and Aspergillus parasiticus areweak pathogens of corn, cotton, peanut, and nut crops: their effect islimited to a slight reduction in crop yield. However, the significantconsequence of crops infected with either of these fungi iscontamination by aflatoxin, which is produced under certain conditionsduring the infection. Traditional control strategies such as breedingcrops for resistance to the fungi or chemical treatments of crops toprevent infection by the fungi have not been effective.

Aflatoxin is a secondary metabolite that appears to be the most potentnaturally occurring carcinogen known (Council for Agricultural Scienceand technology (CAST), 1989). It is suspected of being responsible forthe high incidence of human liver cancer in many areas of the world(Eaton and Gallagher, Ann. Rev. Pharmacol. Toxicol. 34: 135-139 (1994)).Aflatoxin is introduced into the food chain by preharvest andpostharvest contamination of foods and feeds. Also, products fromanimals that have been fed aflatoxin contaminated feed may also becomecontaminated. Currently, the U.S. Food and Drug Administration limitsthe allowable amount of aflatoxin in food to 20 ppb, with slightlyhigher levels allowed in feeds. Because the level of aflatoxin inproducts destined for human consumption is strictly regulated in theU.S., aflatoxin contamination is primarily of economic importance.However, even though aflatoxin levels in foods is limited to 20 ppb, theeffect of chronic exposure to low levels of aflatoxin on human health isunknown. Thus, some European countries require the presence of aflatoxinin foods intended for human consumption to be 0 ppb. In areas of theworld where regulations do not exist, aflatoxin is a serious healthproblem (CAST, 1989).

Approaches to control of aflatoxin have been broadly grouped intopreharvest and postharvest strategies. Proper grain storage can greatlyreduce contamination postharvest, and some decontamination methods,while costly, are used, e.g., ammoniation. However, most researchefforts at control of aflatoxin has been directed at the preharvestelimination of infection and contamination, since the ability to controlpreharvest contamination would reduce the need for postharvestelimination. Preharvest methods have included agricultural practicessuch as irrigation strategies designed to eliminate stress to cropsassociated with drought, which appears to increase production ofaflatoxin by the fungus. Other methods include using regionally adaptedvarieties of crop plants. However, these methods have been expensive toimplement and have not been completely effective. Chemical controlmethods have also been ineffective at controlling infection by thesefungi.

The development of host plants that are resistant to Aspergillusinfection and aflatoxin contamination has not been as successful as haveprograms for breeding resistance to other pathogens. In general, theresistant varieties that have been made are unstable from growing seasonto growing season and from region to region. Also, screening plants forresistance to colonization by Aspergillus spp. and aflatoxincontamination has been difficult. In corn, and frequently in cotton,inoculation methods have been difficult, often requiring wounding theplant to introduce the fungus, which may overwhelm the plants naturalresistance reactions making it difficult to evaluate the plantsresistance mechanisms (Cotty, Plant Dis. 73: 489-492 (1989)).

Methods have been developed for inhibiting mycotoxin production incrops. For example, U.S. Pat. No. 5,942,661 to Keller discloses a methodof inhibiting mycotoxin production by introducing into the plant a geneencoding a lipoxygenase pathway enzyme of the mycotoxin. The method mayproduce transgenic plants that are substantially resistant to mycotoxincontamination. Mycotoxin resistance is further increased by introducinginto the plant antisense genes for the 9-hyperoxide fatty acid producinglipoxygenases. However, reducing aflatoxin contamination by makingtransgenic plants resistant to aflatoxin production is expensive andtime consuming, and since transformation efficiencies varies from plantspecies to plant species, the method may not be successful for all plantspecies. Furthermore, the long-term effect of introducing transgenicplants into the environment is unknown.

Since traditional methods for controlling fungal infection and/orproduction of aflatoxin by breeding, chemicals, or transgenic plantshave not been completely effective, there is a need for an inexpensiveand effective method for either controlling infection of crops by fungisuch as Aspergillus spp. or Gibberella spp., or controlling thebiosynthesis and accumulation of mycotoxins such as aflatoxin ordeoxynivalenol in plants infected with fungi such as Aspergillus spp orGibberella spp., respectively. There is also a need for a rapid andinexpensive method for identification of chemicals or compounds innatural extracts that inhibit production of mycotoxins such as aflatoxinand deoxynivalenol.

SUMMARY OF THE INVENTION

The present invention provides a substantially pure alkaloid compoundthat inhibits the biosynthesis of particular products of secondarymetabolism. In particular, the present invention provides an alkenylpiperidine amide, which can be isolated from Piper nigrum. The alkenylpiperidine amide inhibits transcription from the nor-1 promoter, thetri5 promoter, ver-1 promoter, the verA promoter, the omt-1 promoter,the fas-1a promoter, alfR promoter, the ipnA promoter, and mutantthereof. In a preferred embodiment the alkenyl piperidine amide inhibitsat least transcription of the nor-1 promoter of Aspergillus parasiticus,the tri5 promoter of Gibberella pulicaris or the ver-1 promoter ofAspergillus nidulans without killing the fungus in vitro. In a preferredembodiment, the alkenyl is a C18 alkenyl with one or more double bonds.Preferably, the C18 alkenyl has two to four double bonds.

The compound of the present invention is useful for inhibitingbiosynthesis of a mycotoxin by a fungus growing on a plant material. Inparticular, the compound inhibits the biosynthesis of aflatoxin anddeoxynivalenol. Thus, the present invention provides a formulation whichcomprises as an active ingredient an alkenyl piperidine amide whereinthe alkenyl is a C18 alkenyl with one or more double bonds, or its salt,or its ester, associated with one or more acceptable carriers,excipients or vehicles therefore. Preferably, the C18 alkenyl has two tofour double bonds.

Thus, the present invention provides the use of a compound, which is analkenyl piperidine amide, wherein the alkenyl is a C18 alkenyl with oneor more double bonds for treatment of plant material to inhibitmycotoxin production by a fungus. Further, the present inventionprovides for use of the above compound for the preparation of acomposition for treatment of a plant material to inhibit mycotoxinproduction by a fungus. In particular, wherein the plant material isselected from the group consisting of seeds, nuts, and animal feeds.

The present invention further provides a method for inhibiting mycotoxinbiosynthesis by a fungus in a plant material comprising applying aneffective amount of an alkenyl piperidine amide in a carrier to theplant material wherein the compound inhibits biosynthesis of themycotoxin. In a preferred embodiment, the alkenyl is a C18 alkenyl withone or more double bonds, preferably, two to four double bonds. In themethod, the alkenyl piperidine amide can be provided in the carrier at aconcentration between about 1 and 100 μg/ml. In a preferred application,the plant material i-s selected from the group consisting of seeds,nuts, grains, and animal feeds.

Because the alkaloid of the present invention is able to inhibit thebiosynthesis of mycotoxins, it would be useful to provide transgenicplants that are able to synthesize the alkaloid of the presentinvention. Therefore, the present invention further provides atransgenic plant that contains DNA comprising genes that encode enzymesinvolved in biosynthesis of the alkenyl piperidine amide wherein thecompound synthesized by the transgenic plant inhibits biosynthesis ofthe mycotoxin by the fungus. In a preferred embodiment, transgenic plantproduces an alkenyl piperidine amide wherein the alkenyl is a C18alkenyl with one or more double bonds, preferably, two to four doublebonds.

The present invention further provides a method for identifyingcompounds that inhibit biosynthesis of a product of secondary metabolismsuch as a mycotoxin in a fungus. In particular, a method is provided foridentifying compounds that inhibit biosynthesis of aflatoxin byAspergillus spp. and biosynthesis of deoxynivalenol by Gibberella spp.

Therefore, the present invention provides a method for determiningwhether a compound inhibits biosynthesis of a secondary metabolitecomprising providing a culture of a transgenic fungus comprising areporter gene operably linked to a promoter for a gene involved in thebiosynthesis of the secondary metabolite, providing to the culture thecompound to be determined, incubating the culture containing the extractunder conditions that cause biosynthesis of the secondary metabolite,and measuring expression of the reporter gene wherein absence ofexpression of the reporter gene indicates that the compound inhibitsbiosynthesis of the secondary metabolite.

The present invention further provides a method for identifying andisolating a compound in a material that inhibits the biosynthesis of asecondary metabolite of a fungus comprising providing an extract of thematerial, separating the material into fractions or compounds by achromatography method, providing a spore suspension of a transgenicfungus comprising a reporter gene operatively linked to a promoter thatis the same as the promoter that controls transcription of a geneinvolved in biosynthesis of the secondary metabolite, adding the sporesuspension to the separated compounds, allowing the spores to germinateand grow fungi, and detecting expression of the reporter gene whereinabsence of expression of the reporter gene identifies the fractions orcompounds that inhibits biosynthesis of the secondary metabolite. In apreferred embodiment of the method, the chromatography is thin layerchromatography (TLC) using TLC plates. Preferably, to grow the fungi theTLC plates are incubated in a dark moist atmosphere at 300 C for a timesufficient for the fungi to cover the plate. It is further preferablethat the fungi be lysed by freeze-thawing to release the reporterexpression product.

In a preferred embodiment of the present invention, the secondarymetabolite is a mycotoxin, preferably selected from the group consistingof aflatoxin, deoxynivalenol, or sterigmatocystin. In practicing thepresent invention, it is preferable that the transgenic fungus be afungus selected from the group consisting of Aspergillus parasiticus,Aspergillus nidulans, Aspergillus versicolor, Aspergillus flavus,Gibberella pulicaris, and Gibberella zeae. In the present invention, itis preferable that the reporter gene be operably linked to a promoterthat is selected from the group consisting of nor-1 promoter, ver-1promoter, verA promoter, fas-1a promoter, omt-1 promoter, alfR promoter,ipnA promoter, tri5 promoter, and mutant thereof. In an embodimentfurther still, it is preferable that the reporter gene be selected fromthe group consisting of a gene encoding β-glucuronidase, a gene encodingβ-galactosidase, a gene encoding luciferase, and a gene encodingfluorescence green protein. In an embodiment further still, a transgenicfungus is provided that comprises a reporter gene operatively linked toa constitutive promoter, which provides a control for the method.Preferably, the constitutive promoter is the promoter for the benA geneor mutant thereof.

Therefore, it is an object to provide a compound that inhibitstranscription of one or more genes encoding proteins involved insecondary metabolism of fungi. In particular, compounds that inhibitgenes involved in biosynthesis of mycotoxins.

It is also an object of the present invention to provide a method fordetermining whether an extract comprises compounds that inhibittranscription of one or more genes encoding a protein involved insecondary metabolism of fungi. In particular, compounds that inhibitgenes involved in biosynthesis of mycotoxins.

Further still, it is an object of the present invention to provide amethod for identifying and purifying compounds that inhibittranscription of one or more genes encoding a protein involved insecondary metabolism of fungi. In particular, compounds that inhibitgenes involved in biosynthesis of mycotoxins.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a TLC plate with resolved crude pepper extract were coatedwith Aspergillus parasiticus G5 (nor-1-GUS fusion) in lane A or GAPN2(benA-GUS fusion in lane B. Plates were subsequently coated with the GUSsubstrate X-gluc. The blue areas indicate GUS activity and the whiteareas indicate inhibition of GUS activity by the underlying resolvedcompounds. Fungal growth was seen in the area corresponding to compoundCp2. An unidentified fungitoxic compound inhibited fungal growth in bothstrains (arrow). The TLC solvent front is indicated by the arrowhead.

FIG. 2A shows the effect of various concentrations of Cp2 on aflatoxinbiosynthesis in Aspergillus parasiticus. Samples were taken 48 hoursafter the addition of Cp2 to GMS medium containing the fungus and theaflatoxin components resolved on a TLC plate. The TLC plate wasvisualized under UV light. Lane 1, control; lane 2, extract from fungusgrown in medium containing 2.6 μg/ml Cp2; lane 3, extract from fungusgrown in medium containing 26 μg/ml Cp2; lane 4, extract from fungusgrown in medium containing 39 μg/ml Cp2; lane 5, extract from fungusgrown in medium containing 52 μg/ml Cp2; lane 6, extract from fungusgrown in medium containing 78 μg/ml Cp2.

FIG. 2B shows the effect of various concentrations of Cp2 onaccumulation of nor-1 transcripts in wild-type Aspergillus parasiticusSU-1. The lower panel is an agarose gel of RNA extracted from SU-1 grownat various concentrations of Cp2. The upper panel is a Northern analysisof the same gel probed with a labeled nucleotide probe to the nor-1gene. Lanes 1-3 are samples analyzed 36 hours after Cp2 was added to themedium and lanes 4-8 are samples analyzed 48 hours after Cp2 was addedto the medium. Lane 1, 36 hour control; lane 2, extract from fungusgrown for 36 hours in medium containing 39 μg/ml Cp2; lane 3, extractfrom fungus grown for 36 hours in medium containing 52 μg/ml Cp2; Lane4, 48 hour control; lane 5, extract from fungus grown for 48 hours inmedium containing 2.6 μg/ml Cp2; lane 6, extract from fungus grown for48 hours in medium containing 26 μg/ml Cp2; lane 7, extract from fungusgrown for 48 hours in medium containing 52 μg/ml Cp2.

FIG. 3A shows the effect of Cp2 and piperine on the expression of GUSoperably linked to the ipnA promoter in a transgenic Aspergillusnidulans 48, 72, and 96 hours after addition to the medium. The GUSsubstrate was MUG (4-methylumbelliferyl glucuronide) and the sampleswere visualized using UV light.

FIG. 3B shows the effect of Cp2 and piperine on expression of GUSoperably linked to the tri5 promoter in a transgenic Gibberella zeae 96hours after addition to the medium. The GUS substrate was MUG and thesamples were visualized using UV light.

FIG. 4A shows a schematic diagram of a restriction enzyme map of plasmidpAPGUSN.

FIG. 4B shows a schematic diagram of a restriction enzyme map of plasmidpAPGUSNN. Plasmid pPAPGUSNNA contains the niaD gene in a clockwiseorientation, and pAPGUSSNB contains the niaD gene in a counter-clockwiseorientation.

FIG. 4C shows a schematic diagram of a restriction enzyme map of plasmidpHD6-6.

FIG. 4D shows a schematic diagram of a restriction enzyme map of plasmidpGAP2.

FIG. 5A shows time course assays on the expression of aflatoxinbiosynthesis and GUS activity in parental Aspergillus parasiticus strainC2N.

FIG. 5B shows time course assays on the expression of aflatoxinbiosynthesis and GUS activity in strain G5 containing plasmid pAPGUSN.

FIG. 6 shows GUS expression under the control of the nor-1 promoter inmycelia growing in peanut testa. Aspergillus parasiticus C2N was thefungal strain transformed with pAPGUSN, and the peanut pods comprisingthe testa were stained with X-Gluc.

FIG. 7A shows transformed fungus strain G5 (containing pAPGUSN) grownwithout the addition of a pepper extract. Mycelia were harvested,macerated, and extracts were used to detect GUS activity using the GUSsubstrate MUG. The photograph was taken under UV illumination. Shown arethree independent experiments without the pepper extract.

FIG. 7B shows transformed fungus strain G5 grown with the addition of apepper extract. Mycelia were harvested, macerated, and extracts wereused to detect GUS activity using the GUS substrate MUG. The photographwas taken under UV illumination. Shown are three independent experimentswith the pepper extract.

FIG. 8 shows the genomic DNA of SEQ ID NO:1, which encodes the nor-1gene from Aspergillus parasiticus (GenBank accession M27801). The genespans nucleotides 269-1501 and the exons consists of nucleotides269-317, 424-961, 1020-1119, and 1184-1258.

FIG. 9 shows the genomic DNA of SEQ ID NO:2, which encodes the ver-1gene from Aspergillus parasiticus (GenBank accession M91369). The geneconsists of nucleotides 396-1526 and the exons consist of nucleotides496-822, 873-1196, and 1258-1395.

FIG. 10 shows the cDNA of SEQ ID NO:3, which encodes the omt-1 gene fromAspergillus parasiticus (GenBank accession L22091). The coding regionconsists of nucleotides 12-1268.

FIG. 11 shows the genomic DNA of SEQ ID NO:5, which encodes the aflRgene from Aspergillus parasiticus (GenBank accession L26220). The geneconsists of nucleotides 224-2379 and the coding region consists ofnucleotides 418-1551.

FIG. 12 shows the genomic DNA of SEQ ID NO:6, which encodes the verAgene from Emericella nidulans (GenBank accession L27825). The gene spansnucleotides 555-1449 and consists of three exons, which span 555-887,939-1262, and 1312-1449.

FIG. 13 shows the genomic DNA of SEQ ID NO:7, which encodes the Tri5gene from Gibberella pulicaris (GenBank accession M64348). The genespans nucleotides 401-1612 and consists of two exons, which span 401-869and 930-1612.

FIG. 14 shows the genomic DNA of SEQ ID NO:8, which encodes the benagene from Aspergillus flavus (GenBank accession M38265). The gene spansnucleotides 207-2121 and consists of eight exons, which span 207-218,347-370, 440-466, 578-619, 701-754, 817-1607, 1672-2031, and 2085-2121.

DESCRIPTION OF PREFERRED EMBODIMENTS

All patents., patent applications, and literature references cited inthis specification are hereby incorporated herein by reference in theirentirety. In case of conflict, the present description, includingdefinitions, will control.

Provided herein is a method for determining whether a compound or anextract can inhibit biosynthesis of a secondary metabolite in a fungus.In particular, a method for identifying and purifying compounds thatinhibit transcription of promoters that regulate transcription of genesencoding proteins, enzymes, or regulatory factors that are involved inbiosynthesis of mycotoxins. As demonstrated herein the method isparticularly useful for identifying and isolating compounds that inhibitbiosynthesis of aflatoxin or deoxynivalenol. A novel aspect of themethod is that it enables compounds that inhibit biosynthesis ofmycotoxins to be distinguished and separated from compounds that inhibitgrowth of fungi.

The method disclosed herein enabled identification and isolation of analkaloid from Piper nigrum (pepper), which is an alkenyl piperidineamide wherein the alkenyl group is a C18 alkenyl with two or more doublebonds, that inhibits biosynthesis of aflatoxin and deoxynivalenol. Inone embodiment, the alkaloid compound has the structure

wherein the compound inhibits biosynthesis of a mycotoxin produced by afungus. The position of the double bonds in any one of the species ofthe present invention is determined by the method of Cahoon et al.,Proc. Natl. Acad. Sci. USA 89: 11184-11188 (1992).

The alkaloid of the present invention, designated Cp2, inhibitstranscription from the nor-1 promoter, the ver-1 promoter, the tri5promoter, and the ipnA promoter. These are all promoters for fungusgenes that encode enzymes involved in biosynthesis of secondarymetabolism products. The nor-1 and ver-1 genes encode enzymes involvedin biosynthesis of aflatoxin, the tri5 gene encodes an enzyme involvedin biosynthesis of deoxynivalenol, and ipnA encodes an enzyme involvedin biosynthesis of penicillin. While Cp2 inhibits biosynthesis of theabove secondary metabolites, Cp2 does not inhibit growth of the fungusin vitro. Thus, the inhibitory activity of Cp2 is distinguishable frompiperine, which is also an alkaloid that is isolatable from Pipernigrum, because piperine inhibits fungus growth and transcription fromthe tri5 promoter but not transcription from the ipnA promoter.

Cp2 is useful as an inhibitor of mycotoxin biosynthesis by fungi and,therefore, is useful for preventing the contamination of plant materialwith mycotoxins. Cp2 is particularly useful for preventing contaminationof the plant material with aflatoxin or deoxynivalenol. Plant materialincludes, but is not limited to, grains, nut products such as peanuts,or animal feeds. Cp2 can be isolated from pepper extracts or it can beproduced by chemical synthesis. To prevent mycotoxin biosynthesis in aplant material contaminated with a fungus, Cp2 is admixed with the plantmaterial in an amount sufficient to inhibit the contaminating fungusfrom producing its mycotoxin, in particular, inhibit the fungus fromproducing aflatoxin or deoxynivalenol. A carrier or solution comprisingCp2 can be used in a spray solution for treating the plant material, indry admixture with a carrier that is ingestible, or in a wash solutionfor washing the plant material. The Cp2 concentration in the solutioncan be between about 1 and 100 μg/ml. In culture, Cp2 at a concentrationof about 52 μg/ml or more was shown to completely inhibit transcriptionfrom the nor-1 promoter. Under particular conditions, the concentrationof Cp2 in the solution can be less than 1 μg/ml. Treating the plantmaterial with Cp2 enables the plant material to be stored for anextended period of time with reduced risk that the plant material willbecome contaminated with a mycotoxin. Thus, Cp2, which inhibitsAspergillus spp. and Gibberella spp. from producing aflatoxin anddeoxynivalenol, respectively, when applied to the plant material,reduces the risk that stored plant material will be contaminated withaflatoxin or deoxynivalenol. Furthermore, it has been reported that invivo mycotoxins perform an essential role in the ability of the fungusin invading and colonizing plant tissues. Thus, the present inventionnot only inhibits mycotoxin biosynthesis but can further prevent fungalgrowth on the plant material. The Cp2 is particularly useful inhibitorof mycotoxin and fungal growth in vivo because it inhibits transcriptionof several of the genes involved in mycotoxin biosynthesissimultaneously. The simultaneous inhibition of transcription of severalgenes indicates that fungi may be less able to mutate around theinhibitory effect of Cp2 than they would be in the case of an inhibitordirected against a single gene target. Thus, Cp2 can be used to preventmycotoxin contamination of plant material with a reduced risk thatfungus mutants would arise that are resistant to Cp2 than with mycotoxininhibitors that are directed against a single gene or gene product.

The genes encoding the enzymes involved in Cp2 biosynthesis can beisolated and used to produce transgenic plants wherein the Cp2 isproduced in the seed, nut, or grain of the plant for control ofmycotoxin biosynthesis by fungi growing on the seeds, nuts, or grain.These genes can also be used to transform commercial strains of fungusto control the synthesis of undesirable secondary metabolites inpharmaceutical or food fermentations. These genes can also be used totransform bacteria to enable biosynthesis of Cp2 by commercialfermentation methods.

As demonstrated by the identification and isolation of Cp2, the presentinvention provides a bioassay for identifying extracts and particularcompounds within the extract that inhibit the biosynthesis of mycotoxinsthat include, but are limited to, aflatoxin, sterigmatocystin, ordeoxynivalenol. In a preferred embodiment, chromatography is used toseparate the compounds in the extract, which are then tested asdisclosed herein for the ability to inhibit a promoter for a geneinvolved in the biosynthesis of the mycotoxin. In particular, a promoterselected from the group consisting of the nor-1 promoter, the ver-1promoter, the verA promoter, the fas-1a promoter, the omt-1 promoter,the alfR promoter, the ipnA promoter, and the tri5 promoter.

To determine whether an extract contains at least one compound thatinhibits mycotoxin biosynthesis in a fungus, or to identify and purifythe inhibitory compound, a transgenic fungus is provided, whichcomprises a reporter gene operably linked to a promoter to a geneinvolved in mycotoxin biosynthesis. The transgenic fungus is grown withthe extract in a culture under conditions that stimulate biosynthesis ofthe mycotoxin. Optionally, the method provides that several cultures areprovided, each containing a transgenic fungus with one of theaforementioned promoters operably linked to a reporter gene. It ispreferable that a control culture is also provided, which contains thesame transgenic fungus grown in the absence of the extract, and controlcultures that contain a transgenic fungus comprising a reporter geneoperably linked to a promoter involved in primary metabolism, grown incultures both with the extract and without the extract. When an extractcontains a compound that inhibits mycotoxin biosynthesis, transcriptionof the reporter gene is inhibited when the transgenic fungus is grownwith the extract but is not inhibited when grown in the absence of theextract. In contrast, the control transgenic fungus containing thereporter gene operably linked to a primary metabolism promoter is notinhibited by the extract. Determination of whether transcription of thereporter gene is inhibited or not is accomplished by adding an indicatorsubstrate that enables detection of reporter activity. Preferably, thefungi are lysed prior to addition of the indicator substrate. A suitablemethod for lysing the fungi is freeze-thawing to break down the cellmembranes to allow the reporter to leak out of the fungi. Alternatively,the fungi can be harvested and protein extracts made by methods wellknown in the art, and the protein extracts tested for reporter activity.

To further identify and isolate a compound in an extract that inhibits apromoter controlling transcription of a gene involved in biosynthesis ofa mycotoxin, the compounds of the extract are separated into compounds,components, or fractions by a method such as column or thin-layerchromatography (TLC) chromatography. Preferably, the chromatographymethod is thin layer chromatography and separation of the extracts intocompounds, fractions, or components is accomplished using variousdeveloping solvents, which are well known in the art. The separatedcompounds, fractions, or components are incubated as above with thetransgenic fungus comprising a reporter gene operably linked to apromoter involved in mycotoxin biosynthesis under conditions thatstimulate biosynthesis of the mycotoxin. A control transgenic funguscomprising a reporter gene operably linked to a promoter involved inprimary metabolism is also provided in a control incubation. Inhibitionof transcription of the reporter gene controlled by the mycotoxinpromoter but not the primary metabolism promoter indicates that thecompound, fraction, or component inhibits mycotoxin biosynthesis in thefungus. Inhibition of reporter transcription is determined by detectingreporter activity using an indicator substrate. By using the abovemethod or combining the above method with other purification methodswell known in the art, the inhibitory compound can be purified.Alternatively, the extract is resolved into compounds, fractions, orcomponents by TLC. The TLC plates are then completely coated with aspore solution of a transgenic fungi containing the reporter geneoperably linked to a promoter involved in mycotoxin biosynthesis in amolten agarose solution. The agarose immobilizes the spores on the plateand when the fungi germinates from the spores the agarose preventscross-contamination between particular areas of the plate. Afterallowing the agarose to solidify, the plates are incubated underconditions that promote fungus growth. When fungus growth becomesmanifest, the fungi coated plates are treated to lyse the fungi,preferably by freeze-thawing the fungi. The TLC plates are then treatedwith an indicator substrate that enables determination of whethertranscription of the reporter has been inhibited. Preferably, theindicator substrate is applied as a solution that contains moltenagarose. The agarose, after it solidifies, immobilizes the indicatorsubstrate to prevent cross-contamination.

In a preferred embodiment, the method screens for compounds that inhibitpromoters involved in mycotoxin biosynthesis, which include, but are notlimited to, promoters for genes encoding the following enzymes involvedin the biosynthesis of aflatoxin: nor-1 (FIG. 8; SEQ ID NO:1) fromAspergillus parasiticus (Chang et al., Curr. Genet. 21: 231-233 (1992);Trail et al., Appl. Environ. Microbiol. 60: 4078-4085 (1994)), ver-1(FIG. 9; SEQ ID NO:2) from Aspergillus parasiticus (Skory et al., Appl.Environ. Microbiol. 58: 3527-3537 (1992)), omt-1 (FIG. 10; SEQ ID NO:3)from Aspergillus parasiticus (Yu et al., Appl. Environ. Microbiol. 59:3564-3571 (1993)), fas-1A (SEQ ID NO:4) from Aspergillus parasiticus(Mahanti et al., Appl. Environ. Microbiol. 62: 191-195 (1996)), alfR(FIG. 11; SEQ ID NO:5) from Aspergillus parasiticus (Chang et al., Appl.Environ. Microbiol. 59: 3273-3279 (1993); Payne et al., Appl. Environ.Microbiol. 59: 156-162 (1993)), verA (FIG. 12; SEQ ID NO:6) fromEmericella nidulans (Keller et al., Phytopathol. 84: 483-488 (1994); ordeoxynivalenol such as tri5 (FIG. 13; SEQ ID NO:7) from Gibberellapulicaris (Holn et al., Molec. Plant-Microbe Interactions 5: 249-256(1992)). In a preferred embodiment, the transgenic fungus comprises areporter gene that is operably linked to the nor-1 promoter, which isinvolved in the conversion of norsolorinic acid to averantin in theaflatoxin biosynthesis pathway, or the reporter gene is operably linkedto the promoter for the tri5 gene, which is involved in the biosynthesisof deoxynivalenol. In addition, it is preferable that the method furtherinclude a control transgenic fungus comprising a reporter gene that isoperatively linked to a promoter for a gene known not to be involved insecondary metabolism. An example of such a promoter is the promoter forthe benA gene (FIG. 14; SEQ ID NO:8), which encodes β-tubulin inAspergillus flavus (Woloshuk et al., Appl. Environ. Microbiol. 60:670-676 (1994)). For any one of the above promoters and gene sequences,the present invention includes mutants thereof.

As used herein, the phrase “operably linked to a promoter” refers toboth a recombinant nucleic acid molecule wherein the reporter gene isdirectly linked to the promoter with little or no nucleotides betweenthe promoter and the reporter gene and to a recombinant nucleic acidmolecule wherein the reporter gene is inserted into a gene controlled bythe promoter in the proper codon reading frame to produce a chimeric orfusion polypeptide comprising the promoter's gene product with thereporter polypeptide inserted therein. The advantage of the latter isthat it facilitates generation of transgenic fungi with the reportergene inserted into the promoter's gene in the fungi by double crossoveror homologous recombination. In general, when making plasmid constructscontaining a reporter gene for use as vectors for making transgenicfungi by double crossover, it is preferable that a deletion be made inthe coding region of the promoter's gene and replacing the reporter genewith the deleted material.

In particular embodiments, the fungi used to make the transgenic fungiinclude, but are not limited to, Aspergillus spp. such as Aspergillusparasiticus, Aspergillus nidulans, Aspergillus versicolor, Aspergillusflavus, and Gibberella ssp. such as Gibberella zeae and Gibberellapulicaris. Methods for DNA transformation of fungi have been taught bySkory et al. Appl. Environ. Microbiol. 56: 3315-3320 (1990); Oakley etal., Gene 61: 385-399 (1987); and, a method is taught herein. It is alsoenvisioned that the above assays can be performed using bacteriatransformed with the any one of the aforementioned promoters operablylinked to a gene encoding a reporter.

In particular embodiments, the reporter gene that is operably linked tothe secondary or primary metabolite promoter, includes but is notlimited to, the uidA gene, which encodes β-glucuronidase (GUS); the lacZgene, which encodes β-galactosidase; the luc gene, which encodes fireflyluciferase; the Rluc gene which encodes the Renilla luciferase; or thegene encoding the fluorescent green protein, which is disclosed in U.S.Pat. No. 5,958,713 to Thastrup et al. These reporter genes arecommercially available and methods for their cloning and use are wellknown in the art. In the embodiment demonstrated herein, the reportergene encodes the GUS enzyme. GUS was used as a reporter gene because GUSactivity is easily monitored with a variety of indicator substratesincluding the histological stain,5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), which in thepresence of the GUS enzyme is converted to a blue pigment, and4-methylumbelliferyl-B-glucuronide (MUG), which in the presence of GUScan be converted to a fluorescent compound. These assays are well-knownin the art. The transgenic fungi herein comprise fungi wherein the GUSgene is operably linked to a promoter selected from the group consistingof the nor-1 promoter (isolatable from SEQ ID NO:1), the ver-1 promoter(isolatable from SEQ ID NO:2), the benA promoter (isolatable from SEQ IDNO:8), or the tri5 promoter (isolatable from SEQ ID NO:7) (Trail et al.,Proc. Am. Phytopathol. Soc. Nat. Mtg., Albuquerque, N. Mex. 1994; Payneet al., Appl. Environ. Microbiol. 59: 156-162 (1993)). The abovesequences are particularly suitable for making transgenic fungi bydouble crossover or homologous recombination that produce the geneproduct and reporter as a chimeric or fusion polypeptide.

Examples of the GUS reporter gene operably linked to a promoter areshown in FIGS. 4A-4D which show DNA plasmid vectors pAPGUSN, pPAGUSNN,pHD6-6 (Wu et al., Proc. Curr. Issues Food Safety, National Food SafetyToxicology Center, Michigan State University, East Lansing, Mich.), andpGAP2 (Woloshuk et al., Appl. Environ. Microbiol. 60: 670-676 (1994)),respectively. The above plasmid vectors have been used to transformAspergillus parasiticus strain NR1 (niaD-mutant from ATCC 5862, afl+,disclosed in Chang et al., Curr. Genet. 21: 231-233 (1992)), strain NR2,or strain C2N (Trail et al., Appl. Environ. Microbiol. 60: 4078-4085(1994)). While strains NR1 and C2N were used to demonstrate practice ofthe present invention, other fungal strains, transformed with theplasmid vectors disclosed herein or plasmid vectors containing otherpromoters involved in biosynthesis of secondary metabolites operablylinked to a gene encoding a reporter such as the GUS gene, can be usedto detect inhibitors of secondary metabolites according to the presentinvention.

Plasmid pAPGUSN (FIG. 4A) contains the GUS gene operably linked to thenor-1 promoter at its 5′ end and the nor-1 transcription terminator atits ₃′ end. pAPGUSN was stablely integrated into the genome of C2N, anor-1, niaD+strain, by double crossover insertion. In the same manner asabove, a second plasmid, pAPGUSNN, comprising the nor-1 promoteroperably linked to GUS and a functional niaD gene (FIG. 4B) was stablelyintegrated into strain NR1, which restored the niaD+phenotype to strainNR1, and preserved aflatoxin biosynthesis. A third plasmid similar topAPGUSNN was made with the ver-1 gene promoter operably linked to theGUS gene (plasmid pHD6-6) (FIG. 4C) and used to transform Aspergillusparasiticus. In addition to the above transgenic fungi, a transgenicGibberella zeae expressing GUS controlled by the tri5 gene promoter, atransgenic Aspergillus nidulans expressing GUS controlled by the ipnApromoter, and a transgenic Aspergillus parasiticus expressing GUScontrolled by the benA promoter (plasmid pGAP2 in FIG. 4D) were alsomade in the same manner. The method for making plasmid FLIRT comprisingthe ipnA promoter is disclosed by Bergh et al. in the J. Bacteriol. 178:3908-3916 (1996). Methods for transforming fungi and recovering stabletransformants are well known in the art. The aforementioned transgenicfungi can each be used in the present invention to identify and isolatecompounds that inhibit biosynthesis of mycotoxins.

While particular plasmid vectors for making the transformed fungi aredisclosed herein, transformed fungi equivalent to the transformed fungidisclosed herein can be made using any plasmid that contains the same orsubstantially the same sequences as disclosed by the particular plasmidvectors herein. Methods for isolating, cloning and manipulating DNA arewell known in the are as are methods for producing transgenic fungi.Therefore, the DNA fragments comprising promoter and termination regionsfor any one of the mycotoxin biosynthesis genes can be isolated using ina polymerase chain reaction (PCR) appropriate primers to amplify thepromoter and termination regions. Primers can be designed using methodswell known in the arts. These amplified DNA fragments are then ligatedto the 5′ and 3′ ends of a DNA encoding a reporter to form a cassette.DNA encoding reporters are commercially available. The cassette isinserted into any commercially available plasmid, which is linearizedand used to transform any fungus, including those taught herein. Priorto transformation, additional genes such as the niaD gene can also beinserted into the plasmid. The novelty of the method resides in itsability to identify inhibitors of particular promoters controllingparticular enzymes involved in mycotoxin biosynthesis and to distinguishthe particular inhibitors from inhibitors that affect growth of thefungi, not in the particular transgenic fungi disclosed herein or theparticular plasmids disclosed herein that were used to construct thetransgenic fungi.

It is known that a wide variety of substances can inhibit aflatoxinbiosynthesis; however, many of these substances also inhibit fungalgrowth, and often, the two processes have not been distinguished.However, unlike prior methods for identifying mycotoxin inhibitors, thepresent invention enables compounds that inhibit mycotoxin biosynthesisto be distinguished from compounds that inhibit both mycotoxinbiosynthesis and primary metabolism. Hikoto et al. (Mycopathologia 66:161-167 (1978)) reported that extracts of various condiments and herbaldrugs inhibit mycotoxin biosynthesis. It is well known that peppercontains piperine, an alkaloid that inhibits fungus growth and thereformycotoxin biosynthesis. However, using the method disclosed herein anovel compound in pepper extracts was identified that inhibits aflatoxinbiosynthesis but not fungus growth in vitro. In particular, the methodenabled the identification and isolation of compound Cp2, a novelalkaloid that inhibits the nor-1 promoter and thus, aflatoxinbiosynthesis, but does not inhibit primary metabolism, since growth ofthe fungus was not inhibited in vitro. However, because mycotoxins havean important role in enabling fungi such as Aspergillus ssp. andGibberella ssp. to invade and colonize plant tissues, providing Cp2 to aplant material can effectively prevent the fungi from growing in theplant material.

Identification of Cp2 was by the following culture or batch andchromatography methods of the present invention. Transgenic fungusstrain G5 containing the nor-1 promoter operably linked to the GUSreporter gene was grown in PMS medium, which does not support aflatoxinbiosynthesis. After 72 hours, the transgenic fungus was transferred toGMS medium, which is an aflatoxin induction medium (Buchanan et al.,Appl. Environ. Microbiol. 48: 306-310(1984)). The above nutrient shiftavoids differences in mycelial growth rates, which may occur betweentreatments. After the transfer to GMS medium, a chloroform extract ofblack pepper was added to medium. When GUS activity was measured, thepepper extract inhibited expression of GUS activity in transgenic strainG5 (FIG. 7) whereas a control culture of strain G5, to which no pepperextract was added, produced GUS activity (FIG. 7).

To show that the inhibition of GUS activity by the pepper extract wasbecause of inhibition of the nor-1 promoter and not because somecomponent of the pepper extract inhibited the GUS enzyme itself, theassay was repeated with transgenic fungus containing pGAP2, pAPGUSN(transformant G5), or pAPGUSNN. The pepper extract inhibited expressionof GUS activity when GUS was under the control of the nor-1 promoter(pAPGUSN or pPAGUSNN), but not when GUS was under the control of thebenA promoter (pGAP2). This indicated that the pepper extract did notinhibit the GUS enzyme by binding to the enzyme. Instead, inhibition wasdue to a component of the pepper extract, later identified as Cp2,interacting with the nor-1 promoter. The effect of black pepper extracton expression of GUS activity in Aspergillus transformed with the abovevectors is shown in Table 1. TABLE 1 GUS activity plasmid (promoter)Pepper extract No extract pGAP2 (benA) + + pAPGUSN (nor-1) − + pAPGUSNN(nor-1) − +

The method further enabled the novel Cp2 compound in the pepper extractthat inhibited transcription of the nor-1 promoter, but nottranscription of primary metabolism promoters, to be isolated. Toisolate the Cp2 compound from the pepper extract, an aliquot of thepepper extract was resolved on silica plates by thin-layerchromatography (TLC) The TLC plates were then completely coated with aspore solution of transgenic fungus strain G5 containing about 0.3%molten agarose. After the agarose solidified, the plates were incubatedunder conditions that promoted growth of the transgenic fungus.Preferably, the plates were incubated at 300 C for about two days in thedark. After fungus growth became manifest, the fungi covering the plateswere lysed by freeze-thawing, preferably by freezing at −800 C andthawing at room temperature. Next, the plates were covered with asolution of X-Gluc in about 0.6% molten agarose. The plates were thenincubated 370 C and monitored for β-glucuronidase activity. Blue areassignified that the hyphae in the area had β-glucuronidase activitywhereas non-blue areas indicated the hyphae did not produceβ-glucuronidase due to the presence of underlying inhibitory compounds.One area, designated Cp2, was subsequently shown to contain the novelCp2 of the present invention. Control plates covered with a controlfungus containing GUS under control of the benA promoter indicated thatCp2 did not inhibit fungus growth in vitro, only mycotoxin biosynthesis.

A preparative quantity of pepper extract was then resolved by flashcolumn chromatography and column fractions were collected. The fractionswere each analyzed by TLC as set forth above to identify thosechromatography fractions that contained Cp2. The areas of the TLC platescorresponding to Cp2 were removed from the plate, pooled, concentrated,and re-chromatographed on TLC plates using a second solvent system toresolve the compounds. Optionally, the TLC purification method can berepeated until the compound is purified free of other compounds. Afterany purification step, an aliquot of the inhibitory material can beassayed on TLC plates or in the batch method to ensure that the materialbeing purified retains the ability to inhibit mycotoxin biosynthesis.Purification of Cp2 required an additional TLC chromatography using athird solvent system which resulted in a Cp2 preparation of about 97%purity. As alternatives to TLC, column chromatography (e.g., ionexchange chromatography, size exclusion chromatography, HPLC, FPLC,etc.) or high voltage paper electrophoresis can be used to purifyinhibitory compounds.

Cp2 was determined to inhibit transcription not only from the nor-1promoter but also from the ver-1 promoter, the tri5 promoter, and theipnA promoter. The inhibitory activity of Cp2 is distinguishable frompiperine. Using the batch method of the present invention, piperine wasunable to inhibit transcription from the ipnA promoter (FIG. 3A) eventhough it inhibited transcription from the tri5 promoter (FIG. 3B).Also, piperine inhibits fungus growth in vitro whereas Cp2 does not.Furthermore, Cp2 is distinguishable from other compounds that inhibitaflatoxin accumulation. For example, Huang et al. (Phytopathol. 87:622-627 (1997)) identified from seeds of corn inbred for resistance toaflatoxin accumulation, a protein that inhibited aflatoxin accumulationby inhibiting growth of Aspergillus flavus, and another proteininhibited aflatoxin accumulation with little effect on fungal growth;Russin et al. (Phytopathol. 87: 529-533 (1997)) identified a componentof unknown identity in the wax of kernels of corn bred to be resistantto Aspergillus flavus that is not present in the wax of corn kernelsfrom corn not resistant to Aspergillus flavus; and Ghosh et al. (J.Stored Products Res. 32: 339-343 (1996)) identified three proteins inextracts of sorghum seeds that completely inhibited germination ofAspergillus flavus spores.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

The method for identifying compounds that inhibit mycotoxin biosynthesisis illustrated using ground black pepper (Piper nigrum) extracts andassaying for compounds that inhibit aflatoxin biosynthesis.

Ground black pepper (500 mg) was extracted in 2 L cyclohexanol overnightunder a fume hood with constant stirring. After 24 hours, the mixturewas filtered through Whatman paper and the extracted pepper filtrate wasremoved and in a similar manner to the above, extracted with chloroformfollowed by absolute ethanol. The filtrates from each extraction wereconcentrated by rotary evaporation and analyzed by thin layerchromatography (TLC).

The extracts were separated on TLC plates using a solvent systemconsisting of chloroform:acetone:toluene (25:40:35 v:v:v). Afterwards,the TLC plates were dried in a fume hood to evaporate any solvents lefton the plates, and then placed silica-side down on a UV transilluminatorand UV absorbent spots were traced on an acetate sheet. To identifycompounds with aflatoxin inhibitory properties, a bioassay was inventedthat modified the screening method of Homans et al. (J. Chromato., 51:327-329 (1970)). The bioassay used two transgenic strains of Aspergillusparasiticus. The strain G5 had its nor-1 gene replaced by a DNAconstruct comprising the nor-1 promoter operably linked to GUS reportergene (Xu et al. Physiol. Molec. Plant Pathol. 56: 185-196 (2000)). Thestrain GAPN2 had its niaD gene replaced by a DNA construct containingthe β-tubulin bena promoter operably linked to the GUS reporter gene.

To perform the TLC bioassay, a spore solution of a transgenicAspergillus parasiticus (strain G5) that contained the aflatoxinbiosynthesis promoter nor-1 operably linked to the uidA gene, whichencodes β-glucuronidase (GUS), was made at a final concentration ofabout 1×10⁶ spores per ml in YES medium (a yeast extract and sucrosemedium that induces aflatoxin biosynthesis in Aspergillus spp.)containing 0.3% agarose at 55 to 60° C. (20 ml will cover a plate thathas a surface area of 200 cm²). The spore solution was evenly sprayedacross the TLC plate inclined at a 60° angle in a sterile hood using aglass TLC spray apparatus with care taken to ensure an even coating.After the agarose solidified, the plate was balanced on two glass testtubes on dampened paper towels in a moist chamber assembled from aplastic storage container with a loose lid that was completely linedwith plastic wrap. Plates were incubated for 2 days at 300 C in thedark. Following two days of growth, the plates were frozen for one hourat −80° C. and then thawed at room temperature for at least 30 minutesto break down the cell membranes of the fungi and allow the GUS enzymeto leak out of the fungi. Areas of inhibition of fungal growth weretraced on acetate sheets for comparison to the UV absorbent spots. Thenthe plates were inclined at a 60° angle and sprayed using a TLC platesprayer with a mixture of 15 ml of 2× X-Gluc buffer (100 mM KPO₄, pH7.0, 0.3% K ferricyanide, 0.1% X-Gluc(5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt(Gold Biotechnology, Inc., St. Louis, Mo.) added to 15 ml of 0.6%agarose at 55 to 60° C. immediately before spraying. The plates werewrapped in plastic film and incubated in a plastic storage container at370 C overnight. Areas that lacked β-glucuronidase activity were tracedonto acetate sheets and compared to the locations for the UV absorbentspots and the areas that inhibited fungal growth.

Visual examination showed that the TLC plates were evenly covered withmycelia except for one area indicating that this area contained acompound that inhibited growth of the transgenic fungus (FIG. 1). GUSactivity, which was indicated by a blue color, was not uniform over theentire surface of the TLC plate (FIG. 1, lane A). Additional area thatlacked a blue color but had mycelial growth represented compounds thatwere inhibitory to the nor-1 promoter. Inhibition of GUS activity wasnot detected in a control consisting of a transgenic Aspergillusparasiticus containing plasmid pGAP2 comprising the GUS gene operativelylinked to the promoter for the benA gene which encodes β-tubulin (FIG.1, lane B). The control showed that the inhibitory effect of thecompounds was not due to inhibition of GUS activity but was inhibitionof GUS transcription via the nor-1 promoter. The area displayinginhibition of GUS transcription was chosen and the inhibitory compoundwas isolated.

This example shows that the present invention provides an easy andreliable TLC-based method to detect compounds inhibitory totranscription of secondary metabolism genes in Aspergillus parasiticus.

EXAMPLE 2

The following is a procedure for isolating Cp2 from ground pepperextracts.

Black ground pepper was suspended in cyclohexane (1:2 w:v) with constantstirring overnight. The mixture was filtered and then reextracted withcyclohexane for an additional four hours. The filtrates were combinedand concentrated by rotary evaporation under vacuum at 40° C. Afterrotary evaporation, the cyclohexane extract was observed to have twophases. These phases were separated yielding a phase readily soluble incyclohexane and a less dense phase readily soluble in ethanol. Bothphases were tested to determine which phase contained the bioactivecompound indicated in Example 1. An amount consisting of 10 μl of bothphases were loaded onto separate TLC plates and the plates weredeveloped using a solvent system consisting ofchloroform:toluene:acetone (25-40:35 v:v:v). After development, theplates were dried overnight. Then the GUS bioassay was performed asshown in Example 1. The bioactive compound Cp2 was observed to bepresent in the ethanol soluble phase.

Flash chromatography was used to purify Cp2 from the pepper extractaccording to the method described by Still et al., J. Org. Chem. 43:2923-2925 (1978). Flash chromatography uses an air pressure drivencolumn which has been optimized for fast separations. The column used topurify Cp2 was dry packed with 6 inches of silica gel (grade 9385,230-400 mesh, 60 angstrom available from Aldrich, Milwaukee, Wis.)between two layers of 50 mesh sand. Concentrated pepper extractfiltrates made according to above were applied to the column and thecolumn was then developed using a solvent system consisting ofchloroform:toluene:acetone (25:40:6 v:v:v). Five ml aliquots werecollected and analyzed on TLC plates to identify Cp2. The fractionscontaining Cp2 were pooled, concentrated by rotary evaporation, andresuspended in 100 proof ethanol and stored under refrigeration at 4° C.Approximately 10 mg of the concentrated fraction containing Cp2 wasloaded onto a 20×20 cm preparatory TLC plate containing 1000 μm silicagel (60 angstrom, available from Whatman, Clinton, N.H.). The TLC platewas developed three times, each time using a solvent system consistingof hexane:acetone (2:1 v:v) After the third development, the TLC platewas dried and assayed for GUS inhibition as in Example 1. The bioactiveband containing Cp2 was scraped out of the silica plate and eluted fromthe silica using chloroform:ethanol (4:1 v:v). The Cp2 product was thenconcentrated by gaseous hydrogen gas and loaded onto a TLC plate asabove and developed three times, each time with a solvent systemconsisting of hexane:ethyl acetate (4:1 v:v) which provided sufficientseparation to enable a single band containing Cp2 to be located. The Cp2was recovered with a purity of approximately 97%. TLC bioassays as abovewere then used to confirm the inhibitory properties of the nearlyhomogenous preparation of Cp2. A preliminary structure of Cp2 wasdetermined by mass spectrometry to comprise an unsaturated C18 fattyacid amide of piperidine.

Therefore, this example shows the isolation of the aflatoxin inhibitorycompound of the present invention, Cp2, which had been identified by themethod of the present invention demonstrated in Example 1.

EXAMPLE 3

Cp2 has a demonstrable effect on aflatoxin biosynthesis and nor-1transcription. This example used Aspergillus parasiticus SU-1, awild-type aflatoxin producing isolate, and consisted of analyzingaflatoxin biosynthesis by TLC and aflatoxin mRNA transcription byNorthern analysis.

A nutritional shift protocol (Skory et al., Appl. Environ. Microbiol.56: 3315-3320 (1990)) was used with the modifications below to determinethe effect of Cp2 on expression of the nor-1 gene and aflatoxinbiosynthesis. Six cultures of fungi were incubated in PMS (peptonemineral salts), a medium that does not induce biosynthesis of aflatoxin.After 48 hours, the mycelium from each culture were separatelytransferred to GMS (glucose mineral salts), a medium that inducesbiosynthesis of aflatoxin. At the same time Cp2 was added to five of thesix cultures to provide cultures having a final concentration of 2.6μg/ml, 26 μg/ml, 39 μg/ml, 52 μg/ml, and 78 μg/ml, respectively.Thirty-six hours later, a sample of each culture was removed forNorthern analysis. Forty-eight hours later, the culture filtrate wasanalyzed by TLC for aflatoxin biosynthesis and RNA was extracted fromthe mycelium for Northern analysis. Direct competitive ELISA analyseswere also performed to determine whether, for each sample, aflatoxin B1(AFB1) was in the medium. The procedure was performed as described byPeska (J. Assoc. Off. Anal. Chem. 71: 1075-1081 (1988)) with anti-AFB1antibodies and AFB1-horseradish peroxidase conjugate (both available byname from Michigan State University, East Lansing, Mich.).

FIG. 2A shows Cp2 caused a reduction in the amounts of aflatoxins B1 andG1. FIG. 2A further shows that Cp2 at a concentration greater than 26μg/ml appeared to completely inhibit biosynthesis of aflatoxin.Interestingly, Cp2 also caused an increase in the amount of anunidentified pigment that correlated with the decrease in aflatoxin. Thesignificance of the increase in this pigment is unknown. FIG. 2B showsthat Cp2 had an effect on the accumulation of nor-1 transcripts in thewild-type isolate SU-1. Samples had been collected 36 hours and 48 hoursafter adding the Cp2. FIG. 2B shows that Cp2 at a final concentration of52 μg/ml appeared to completely inhibit nor-1 transcription within 36hours after addition to the medium (lane 3). Lower concentrations of Cp2did not completely inhibit nor-1 transcription (lanes 2, 5, 6 and 7).

This example demonstrates that Cp2 inhibits transcription of the nor-1gene in Aspergillus parasiticus but does not inhibit growth ofAspergillus parasiticus itself.

EXAMPLE 4

The effect Cp2 and piperine on other secondary metabolite promoters iscompared. For this comparison several transgenic fungi containing GUSoperably linked to particular promoters involved in secondary metabolismpathways were used. A transgenic strain of Aspergillus nidulans (apenicillin producing fungus), FLIRT with the promoter for the ipnA gene(a gene involved in penicillin biosynthesis) operably linked to the GUSreporter gene, was provided by Dr. A. Brakhage, Technical University ofDarmstädt, Germany and a transgenic strain of Gibberella zeae with thepromoter for the tri5 gene (a gene involved in deoxynivalenolbiosynthesis) operably linked to the GUS reporter gene, was provided byDr. N. Alexander, U.S. Department of Agriculture, Peoria, Ill.

Each transgenic fungus was grown in mycotoxin inducing liquid mediumcontaining Cp2 or piperine. Aliquots were harvested from the cultures48, 72, and 96 hours after the addition of either the Cp2 or piperine.Protein extracts were made from the harvested mycelia, and 10 μg/mlaliquots were tested for GUS activity. The substrate used to measure GUSactivity was the fluorescence compound 4-methylumbelliferyl (MUG), whichfluoresces under UV light.

FIG. 3A shows that ipnA promoter in Aspergillus nidulans was inhibitedby Cp2 but not by piperine whereas FIG. 3B shows that the tri5 promoterin Gibberella zeae was inhibited by both Cp2 and piperine.

The results in this example demonstrate that even though both Cp2 andpiperine are obtainable from pepper, Cp2 and piperine aredistinguishable compounds.

EXAMPLE 5

Construction of plasmid pAPGUSN having the GUS gene operably linked tothe nor-1 promoter is illustrated. Standard molecular biology techniqueswere used to construct pAPGUSN.

A 3.1 kb HindIII-Bsu361 DNA fragment from the nor-1 gene (sequenceavailable from Trail et al., Appl. Environ. Microbiol. 60: 4078-4085(1994) that included the translational start site and 64 nucleotides 3′of the translational start site was blunt-end ligated to a 5′ NcoI siteof a DNA encoding uidA (GUS reporter gene) using a 10 bp HindIII DNAlinker (available from New England Biolabs, Beverly, Mass.) to maintainintegrity of the reading frame. To form the translational terminationsequence, a 2.0 kb BstY1 DNA fragment from the nor-1 3′ untranslatedregion, including 18 bp upstream of the translational termination site,was ligated to the BamHI site at the 3′ end of the GUS gene. The ligatedproduct inserted into an ampicillin resistant plasmid to generateplasmid vector pAPGUSN. The plasmid vector was sequenced at the 5′junction of the nor-1-GUS fusion to confirm that the correct readingframe was preserved using primers for the promoter and for the GUS genefor double-stranded DNA sequencing. Sequencing was performed using aSequenase chain-termination sequencing kit (available United StatesBiochemical Corp., Cleveland, Ohio) according to the manufacturer'sinstructions. FIG. 4A shows the plasmid map for pAPGUSN.

EXAMPLE 6

Aspergillus parasiticus C2N is transformed with pAPGUSN to illustrateconstruction of transgenic fungi that express GUS under the control of asecondary metabolism promoter.

Aspergillus parasiticus C2N is a nor-1 disrupted transformant thataccumulates the aflatoxin precursor, norsolorinic acid (NA). C2N wasderived from NR-1, which is a nitrate reductase-deficient aflatoxinproducing isolate, derived from wild-type Aspergillus parasiticus SU-1(available as NRRL 5862) by spontaneous mutation to chlorate resistance(Horng et al., Molec. Gen. Genet. 224: 294-296 (1990)), by inserting thenitrate reductase gene, niaD, into the nor-1 gene as described in Trailet al. (Appl. Environ. Microbiol. 60: 4078-4085 (1994)). C2N strainsaccumulate the orange-pigmented aflatoxin precursor norsolinic acid andmakes reduced amounts of aflatoxin due to bifurcation in the aflatoxinbiosynthesis pathway. Even though the aflatoxin produced by strain C2Nis reduced in comparison to strain SU-1, the timing of aflatoxinbiosynthesis is similar. Double recombination between the nor-1 flankingregions of pAPGUSN and the nor-1 flanking regions on the chromosomeresults in the replacement of the disrupted nor-1 in C2N with thenor-1-GUS fusion, resulting in a norsolorinic acid-accumulating, niaD−,GUS+ transformant. This causes the loss of a functional niaD genepresent in the disrupted nor-1 gene of C2N, thus rendering the GUSexpressing transformants to niaD−.

To make the transgenic Aspergillus parasiticus C2N, the plasmid pAPGUSNwas linearized with Kpn1 before transformation. Polyethyleneglycol-mediated transformation was carried out as described in Oakley etal., Gene 61: 385-399 (1987)) with modifications as disclosed in Skoryet al. (Appl. Environ. Microbiol. 56: 3315-3320 (1990)).

Therefore, to perform the fungal transformations, 1×10⁸ conidia wereinoculated into a 250 ml Erlenmeyer flask containing 100 ml Czapek-Dox(CZ) medium (Difco) supplemented with 1% peptone or YES medium. Prior totransformation the flask was coated with a silanizing agent such asPROSIL, or dichlorodimethylsilane to prevent the mycelium from adheringto the glass and growing at the air interface. The culture was grownovernight at 29° C. in an orbital shaker (about 16-18 hours). Growth wasvisibly evident, but not excessive. Ideally, microscopic examinationrevealed that the majority of the conidia had formed germ tubes whichwere beginning to branch. The mycelial growth was harvested using asterile Buchner funnel containing a MIRA-CLOTH filter and washed withwater to remove spores. Then, the collected cells were transferred to asterile 250 ml flask and resuspended in 40 CZ medium. To makeprotoplasts, 40 ml of digestion solution (filter sterilized 5 mg/mlNovozyme-234 in 1.1 M KCL, 0.1 M citrate, pH 5.8) was added. The cellswere incubated for 3 hours at 300 C with gentle shaking. Afterwards, thecells were filtered by gravity through a 29 μm nylon mesh weave filterand the protoplasts harvested at 5,000 rpm in a Sorval SS34 rotor(3,000×g) for 15 minutes at 4° C. The following operations wereperformed on ice. Next, the protoplasts were resuspended in 1 ml PEGbuffer (0.6 M KCL, 0.05 M CaCl₂₁ 10 mM Tris-HCl, pH 7.5) and pelleted ina microcentrifuge at 4,500×g for 1 minute. This washing process wasrepeated three times. Afterwards, the washed protoplasts wereresuspended in no more than 500 μl PEG-buffer and distributed in 100 μlaliquots for transformation. Five μl DNase inhibitor aurentricaboxylicacid (20 mM aurentricarboxylic acid, 5 mM Tris-HCl, pH 7.0) was addedand mixed gently. Then, 1-10 μg of plasmid DNA was dissolved in a volumeof less than 10 μl and added followed by addition of 50 μl of freshlyprepared and filter sterilized PEG solution (25% polyethylene glycol3350, 0.6 M KCL, 0.05 M CaCl₂₁ 10 mM Tris-HCl, pH 7.5). Thetransformation mixture was gently mixed and incubated on ice for 20minutes. Then, 850 μl PEG solution was gently added and the mixtureallowed to sit at room temperature for 30 minutes.

After the transformation reaction, nitrate non-utilizing transformants(niaD−, GUS+) of strain C2N were selected on CZ medium amended with 58g/L potassium chlorate, 100 g/L glutamate, and 20% sucrose (CCGSmedium). Coconut agar medium (CAM; made according to Arseculeratne etal. (Appl. Microbiol. 18: 88-94 (1969)), an aflatoxin inducing medium,was used to screen the transformants for changes in accumulation ofaflatoxin or precursors according to Davis et al. (Microbiol. 53:1593-1595 (1987)).

In two transformation experiments, six of the eight transformantsrecovered from the CCGS selection medium produced GUS activity asmeasured by a MUG assay. Southern analysis confirmed the presence of asingle nor-1-GUS fusion genes at the site of nor-1 and the accompanyingloss of the niaD gene from that region. Three of the GUS expressingtransformants had unexpected rearrangements present in their DNA at thenor-1 region and were eliminated from further analysis. Unexpectedly,replacement of the disrupted nor-1 gene with the nor-1-GUS fusionresulted in the loss or NA accumulation in all cases, although aflatoxinB1 continued to be produced as expected. Further analysis by Southernhybridization did not reveal any explanation for the loss of pigmentproduction, although small rearrangements of nucleotides in the regionof insertion that might affect expression would not have been detectedby Southern hybridizations. Among the three remaining transformants,transformant G5 produced the highest amount of GUS activity in cultureand was chosen for further study.

EXAMPLE 7

Transformants made in Example 5 were analyzed for their growth,aflatoxin biosynthesis, and GUS activity in culture.

Flasks containing 100 ml of YES broth and 5 glass beads to keep themycelia dispersed, were inoculated with 1.75×10⁷ spores each. Thecultures were grown in an orbital shaker at 175 rpm at 28° C. Myceliawas harvested 16, 24, 36, 48, and 72 hours after inoculation. Dryweights were determined by drying the mycelial mats overnight at 60° C.GUS activity assays were performed on 10 mg total protein from groundtissue extracts using the GUS substrate MUG as described in Liang etal., Appl. Environ. Microbiol. 63: 1058-1065 (1997). GUS activity wasdetermined using a spectrofluorometer with an excitation wavelength of365 nm and an emission wavelength of 455 nm. Protein content of thesupernatant was determined by a BCA assay (available from SigmaChemicals, St. Louis, Mo.). Direct competitive ELISA analyses wereperformed on samples of the culture medium to determine concentrationsof aflatoxin B1. The procedures performed as described in Peska (J.Assoc. Off. Anal. Chem. 71: 1075-1081 (1988)) with aflatoxin B1monoclonal antibodies and aflatoxin B1-horseradish peroxidase conjugate.

The results show that there was a similar temporal pattern of aflatoxinB1 biosynthesis between paternal isolate C2N and G5 with lower levels ofbiosynthesis by G5 (FIG. 8). Biosynthesis of aflatoxin B1 in NR1 (niaDparent of C2N) exhibited a similar temporal pattern but, as expected,reached a higher quantity, 42.5 μg/ml culture medium after 72 hours.Comparison of GUS expression by G5 and C2N showed that mycelial extractsof G5 had increased GUS activity for up to 72 hours and no GUSexpression was detected in mycelial extracts of C2N (FIG. 9). Dryweights were not significantly different among all cultures at each timepoint.

EXAMPLE 8

The transgenic fungi were also evaluated for its ability to colonizepeanut plants.

The transgenic fungus was introduced onto peanut plants and the peanutplants were cultivated under drought conditions. About 6-8 weeks afterintroduction, the peanuts were harvested and the harvested podsunderwent treatment for GUS expression. The kernels were split and thehalves were cut perpendicular to their longitudinal axis into piecesapproximately 4 to 5 mm wide. To follow the path of infection of thefungus through the peanut, the correct orientation of the shell,integument, and kernel was maintained during fixation and embedding.This was accomplished by threading a nylon strand through the center ofeach peanut piece, penetrating the shell, integument, and kernelsequentially. The threaded pieces were prepared for cytological study bybriefly fixing with 5% formaldehyde in 50 mM potassium phosphate, pH 7.0for 5-30 minutes on ice under a vacuum. This preliminary fixation killedthe fungus while not affecting GUS activity. After rinsing in steriledistilled water, the peanut parts were incubated overnight with X-Glucin 50 mM potassium phosphate, pH 7.0, containing 0.5 mM potassiumfericyanide and 10 mM EDTA for detection of GUS activity. Following abrief rinse in water, the stained tissues were transferred to a solutionof 3.7% formaldehyde, 5% acetic acid, 47.5% ethanol for at least 24hours. Then, the tissues were dehydrated through a tert-butanol seriesand embedded in paraffin (PARAPLAST PLUS available from FisherScientific, Inc., New Brunswick, N.J.). During the final embedding step,the nylon thread was removed and the peanut pieces were aligned to allowsectioning through all three tissues perpendicular to the original longaxis of the peanut. Paraffin blocks were sectioned at 12-15 mm, andserial sections were placed on glass slides coated with Haupt'ssolution. Following dewaxing of the sections with xylene and rehydrationthrough an ethanol series, some of the sections were stained with 0.2%Chlorazol Black. The sections were mounted in Permount. Microscopy wascarried out on a Ziess Axioskop equipped with DIC optics or an OlympusVanox-S microscope with phase contrast optics.

When the peanuts were stained for GUS using the substrate X-Gluc, thefungus was clearly visible in the infected kernels. However, the bluecolor associated with GUS activity was not observed in conidia,conidiophores, nor the external mycelia surrounding the pod. This wasexpected because aflatoxin precursors do not accumulate in conidia andconidiophores (Keller at al., Phytopathol. 84: 483-488 (1994)). FIG. 5shows that the production of GUS activity in the transgenic fungus wassimilar in time course to aflatoxin biosynthesis. These results indicatethat the nor-1 promoter was functioning in the transgenic fungus in thesame manner as it functions in the wild-type fungus.

A transformant of Aspergillus parasiticus strain NR2, strain 664, wasmade, which contained the GUS reporter gene under the control of thever-1 promoter integrated into the ver-1 gene without disrupting normalaflatoxin biosynthesis (Liang et al., ibid). Strains NR1 and NR2 areindependent spore isolates of the same spontaneous mutant of chlorateresistance.

Transgenic strains G5 and 664 can be used as in the bioassay of thepresent invention to detect and identify compounds that inhibit thebiosynthesis of aflatoxin.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1-12. (Cancelled)
 13. The use of a compound which is an alkenylpiperidine amide wherein the alkenyl is a C18 alkenyl with one or moredouble bonds for treatment of plant material to inhibit mycotoxinproduction by a fungus.
 14. The use of the compound in claim 13 for thepreparation of a composition for treatment of a plant material toinhibit mycotoxin production by a fungus.
 15. The use of the compound inclaim 14 wherein the plant material is selected from the groupconsisting of seeds, nuts, grains, and animal feeds.
 16. A method forinhibiting biosynthesis of a mycotoxin on a plant material comprising:applying an effective amount of a composition comprising an alkenylpiperidine amide in a carrier to the plant material whereby thecomposition inhibits biosynthesis of the mycotoxin.
 17. The method ofclaim 16 wherein the alkenyl is a C18 alkenyl with two or more doublebonds.
 18. The method of claim 16 wherein the C18 alkenyl has two tofour double bonds.
 19. The method of claim 16 wherein the alkenylpiperidine amide in the carrier is at a concentration between about 1and 100 μg/ml.
 20. The method of claim 16 wherein the plant material isselected from the group consisting of seeds, nuts, grains, and animalfeeds. 21-44. (Cancelled)